KR100241184B1 - Angiogenin inhibitor - Google Patents

Abstract

The present invention relates to inhibitors of angiogenin. More specifically, the present invention relates to a peptide having an antagonistic ability against human angiogenin that induces renal angiogenesis developed using a peptide library expressing phage and a method of inhibiting renal angiogenesis using the same. will be. The peptide of the present invention concentration-dependently inhibits the binding between human engiogenin and its receptor actin, and thus inhibits neovascularization induced by human engiogenin without any effect on existing blood vessels. It can be effectively used as a therapeutic agent for diseases such as rheumatoid arthritis and diabetic blindness, etc. in which formation is necessary.

Description

Inhibitors of Engiogenin

The present invention relates to inhibitors of angiogenin. More specifically, the present invention relates to a peptide having an antagonistic ability against human angiogenin that induces renal angiogenesis developed using a peptide library expressing phage and a method of inhibiting renal angiogenesis using the same. will be.

Renal angiogenesis is a term used to describe the formation of new blood vessels in existing blood vessels.In normal conditions such as organ formation or wound healing, angiogenic and anti-angiogenic factors They are controlled by elaborate interactions.

However, in various diseases such as cancer cell growth and metastasis, rheumatoid arthritis and diabetic blindness, renal angiogenesis is indispensable, and renal angiogenesis is not precisely controlled and occurs continuously. Indeed, in many types of human cancers, the number and density of capillaries formed in cancer tissues are known to be closely correlated with the potential for cancer metastasis.

To date, dozens of angiogenic inducers are known to be involved in renal angiogenesis, one of which is angiogenin, a potent angiogenic protein present in vivo (Fett, JW et al. , Biochemistry, 24: 5480 (1985)), and it has been shown through experiments with monoclonal antibodies that human cancer cells secreting them are essential for growth and metastasis in vivo (Olson, KA et al., Proc). Natl.Acad. Sci., USA, 92: 442 (1995). However, the exact mechanism by which angiogenin induces angiogenesis has not been elucidated. However, the binding of angiogenin to a receptor called actin, which exists on the surface of endothelial cells that produce blood vessels, is associated with angiogenesis. (Hu, GF et al., Proc. Natl. Acad. Sci., USA, 90: 1217 (1993)). Therefore, it is expected that a substance that inhibits the binding between angiogenin and actin may not only inhibit angiogenesis induced by angiogenin but also inhibit growth and metastasis of cancer cells secreting angiogenin.

On the other hand, currently used cancer treatments are effective to some extent in the primary cancer (primary cancer), but the types of cancer cells are very diverse, cancer cells become fast resistance to most anti-cancer drugs, once metastases to other organs Metastatic cancer has exposed many limitations to clinical use due to the problem of almost losing its function as a therapeutic agent. Therefore, there is an urgent need for the development of new cancer treatments that can overcome the above-mentioned problems.

Accordingly, the present inventors have diligently researched to develop a neovascularization inhibitor having the above-mentioned problems, that is, diversification of cancer cell types, obtaining resistance to anticancer drugs, and whether cancer cells metastasize before or after their use as anticancer agents. Peptides of specific amino acid sequences developed using expressing peptide libraries (peptide random sequence mixtures) inhibit the binding between human angiogenin and its receptor actin and are induced by human cancer cells that secrete angiogenin and angiogenin Effectively inhibiting angiogenesis, confirming that it can be used as an anticancer agent, the present invention was completed.

After all, the main object of the present invention is to provide a peptide having antagonistic ability against human engiogenin that induces renal angiogenesis.

Another object of the present invention is to provide a method of inhibiting renal angiogenesis by administering the electrical peptide to a mammal in which renal angiogenesis continues abnormally.

1 shows a genetic map of phagemid constituting the peptide library expressing phage.

FIG. 2 is a graph showing that type A and B phages selected from phage libraries specifically bind engiogenin.

FIG. 3 is a graph showing that the binding ability to angiogenin is inhibited by treatment of a reducing agent (1 mM DTT) or protease (Factor Xa) to the A and B phages.

4 is a graph showing that type A and B phage binding to engiogenin is concentration-dependently inhibited by actin, an angiogenin receptor.

5 is a graph showing that the binding of the type A and phage phages to engiogenin is concentration-dependently inhibited by the ANI-E peptide derived from the type A peptide.

FIG. 6 is a graph showing that glutamic acid between disulfide bonds and cysteine of peptides is important in the activity of ANI-E peptides which concentration-dependently inhibits the binding between angiogenin and actin.

FIG. 7 is a graph showing the results of quantitative analysis of the extent to which renal angiogenesis induced by human prostate cancer cells secreting angiogenin is inhibited by the ANI-E peptide.

8 is a graph showing that the ANI-E peptide does not directly affect the growth of human prostate cancer cells.

Hereinafter, the present invention will be described in more detail.

To prepare peptide libraries expressing phage, oligonucleotides of 5′-TG CTG GCC (NNK) ₂ TGC (NNK) ₄ TGC (NNK) ₂ ATA GAA GGA CGC CAT GC-3 ′ and 5′-GGC CGC ATG GCG TCC TTC TAT (MNN) ₂ GCA (MNN) ₄ GCA (MNN) ₂ GGC CAG CT-3 ′ Oligonucleotides, wherein N is A, T, G or C; K is G or T; and M is C or A, respectively) were synthesized, fused, and then introduced into E. coli by inserting phagemid pCANTAB5E (Pharmacia Biotech., USA) digested with restriction enzymes Sfi 1 and Not 1. The phage peptide library was then prepared by culturing E. coli, infecting with M13K07 helper phage, and centrifuging to obtain supernatant.

From the electric phage peptide library, the human phages were bound to and combined with human engineeriogenin through three stages of biopanning using phosphate buffer containing 50 nM, 500 nM and 5 μM of actin and pH 2.2 solution (glycine-HCl). Phage removed by actin known as the receptor of xenin were selected.

The nucleic acid was purified from the selected phage, and the nucleotide sequence thereof was determined. As a result, the nucleotide sequence encoding the type A peptide (Gly-Glu-Cys-Arg-Glu-Asn-Val-Cys-Met-Gly) and The base sequence encoding the type B peptide (Asn-Thr-Cys-Lys-Arg-Phe-Asn-Cys-Asn-Leu) was determined.

Phages expressing these type A and B peptides specifically bind to human engiogenin through an electric peptide, and the treatment of the phage with a reducing agent inhibits the binding ability to human engiogenin. The structure was found to be important for binding. In addition, binding of phage expressing type A and B peptides to human engiogenin was found to be concentration-dependently inhibited by actin, an engiogenin receptor.

Against this background, two cysteine disulfide-linked peptides were chemically synthesized with the following amino acid sequence derived from the amino acid sequence of the A-type peptide:

NH ₂ -Ala-Gln-Leu-Ala-Gly-Glu-Cys-Arg-Glu-Asn-Val-Cys-Met-Gly-Ile-Glu-Gly-Arg-COOH

The electrosynthetic peptide inhibits the binding of phage expressing the A-type peptide to angiogenin and the binding between human angiogenin and its receptor actin, thereby inhibiting the neovascularization induced by human engiogenin. At this time, glutamic acid between the restricted structure by disulfide bond and cysteine is important for the activity of the peptide.

In addition, the electrical peptides inhibit the angiogenin secreted by cancer cells and the like to inhibit angiogenesis induced by cancer cells and the like, but do not affect existing blood vessels, and thus may be usefully used as cancer therapeutic agents and the like without side effects.

Therefore, the angiogenesis can be suppressed by administering the peptide of the present invention to a mammal showing cancer, rheumatoid arthritis, diabetic blindness, and the like, or a mammalian model showing electrical symptoms.

Meanwhile, peptide derivatives prepared by adding site-specific mutations such as substitution, deletion, and addition of amino acids to peptides of the present invention inhibit the binding between human angiogenin and actin of its receptors, and thus do not affect any of the existing blood vessels. As long as it exhibits the activity of inhibiting formation only, it will be said to belong to the scope of the present invention.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

[Preparation of peptide library expressing phage]

To prepare a peptide library expressing phage, first 5′-TG CTG GCC (NNK) ₂ TGC (NNK) ₄ TGC (NNK) ₂ ATA GAA GGA CGC CAT GC-3 ′ (hereinafter, 'OL-52' Oligonucleotides of 5′-GGC CGC ATG GCG TCC TTC TAT (MNN) ₂ GCA (MNN) ₄ GCA (MNN) ₂ GGC CAG CT-3 ′ (hereinafter referred to as 'OL-59' for convenience). Were synthesized and phosphorylated at the 5 'position. Wherein N is A, T, G or C; K is G or T; And M represents C or A, respectively.

8 μg of OL-52 synthesized above and 9 μg of OL-59 were mixed in an aqueous solution containing 1 M sodium chloride, heated at 96 ° C. for 10 minutes, and fused while slowly cooling to room temperature. Meanwhile, phagemid pCANTAB5E (Pharmacia Biotech., USA) was digested with Sfi 1 and Not 1 restriction enzymes and subjected to agarose gel electrophoresis to separate DNA fragments of about 4 kb in size.

20 μg of the isolated phagemid and 10-fold fused OL-52 and OL-59 were mixed and combined with T4 ligase and subjected to 50 electrophoresis and introduced into E. coli strain TG1 cells (Pharmacia Biotech, USA). Escherichia coli cultured with nucleic acid was infected with M13K07 helper phage (Pharmacia Biotech, USA) when the absorbance at 600 nm reached 0.6. The infected E. coli was incubated for one hour, and the culture was centrifuged at 800 x g for 10 minutes to obtain a supernatant. The phage library thus grown was stored in a cold state.

The library prepared above consists of 300 million different phages, and each phage expresses a single kind of peptide. These peptides are linked to the amino terminus of pIII, the skin protein of phage, via the recognition amino acid sequence (Ile-Glu-Gly-Arg) of a specific protease (Ile-Glu-Gly-Arg), as shown in FIG. Is X ₁ X ₂ Cysteine X ₃ X ₄ X ₅ X ₆ Cysteine X ₇ X ₈ . In this case, X means all 20 amino acids. The peptide was prepared such that its structure was limited by disulfide bonds between two cysteines immobilized in the middle.

Example 2

[Selection of Phage Expressing Peptides with Antagonistic Ability to Human Engiogenin]

From the phage library prepared in Example 1, the following three steps of biopaning were performed to select phages that bind to human engiogenin and whose binding is removed by actin known as the receptor for engiogenin. . That is, 100 ng of human engineeriogenin (R & D Systems, USA) was immobilized on a 96-well ELISA plate (Nunc, USA), and phosphate buffer containing 1.5% of albumin was treated at 37 ° C. for 4 hours, and then 10 ^Twelve phage libraries were added and bound with human engiogenin for 2 hours at 37 ° C. Subsequently, each of the phosphate buffer and phosphate buffer containing 0.1% of Tween 20 was washed 10 times to remove phages that did not adhere to engiogenin.

Then, the cells were reacted with phosphate buffer containing 50 nM of actin at 37 ° C. for 1 hour to remove phages attached to angiogenin washed with 50 nM of actin, followed by 1 hour at 37 ° C. with 5 μM of phosphate buffer containing actin. The phages attached to the washed out geogenin were collected and expanded.

6.5 × 10 ¹⁰ of these propagated phages were again bound to engiogenin to remove non-stick phages, and phages washed with phosphate buffer containing 50 nM of actin in the same manner as before, followed by a solution of pH 2.2. Reaction phages were washed with (0.1 M glycine-HCl) for 10 minutes to collect and grow phage.

1.2 × 10 ¹¹ propagated phages were again combined with engiogenin, and the bound phages were washed stepwise as described above with 50 nM, 500 nM and 5 μM of phosphate buffer and pH 2.2 solution, followed by collection of each phage. It was. To determine the number of phage collected at each step, phages were infected with E. coli strain TG1 and the number of individuals resistant to ampicillin was determined.

Table 1 below shows the results of the screening process over the three steps described above.

As shown in Table 1, the yield of phages bound to angiogenin and removed by 5 μM actin increased 5,000 times from 5.7 × 10 ⁻⁹ in one step to 2.8 × 10 ⁻⁵ in three steps, and also in three steps of screening. Phage binding to engiogenin was almost eliminated by 5μM actin, indicating that the screening process was effectively applied.

Therefore, 27 phages washed with 5 μM actin and pH 2.2 solution were randomly grown and propagated in the final three stages of selection, followed by phenol extraction to purify the nucleic acid, and sequenced 5′-TCTGTATGAGGTTTTGCT-3. Was determined using the oligonucleotide of ′, 23 of which phages encode the same type A peptide (Gly-Glu-Cys-Arg-Glu-Asn-Val-Cys-Met-Gly). The two phages showed the nucleotide sequence encoding the type B peptide (Asn-Thr-Cys-Lys-Arg-Phe-Asn-Cys-Asn-Leu), and the bases of the remaining two phages. The sequence could not be determined (see Table 2). Therefore, it was found that phage expressing specific peptides (Type A and B) was selectively amplified from the phage library through the three-step selection process.

Example 3

[Characteristics of Phage Expressing Type A or Type B Peptides]

The phages expressing the A-type peptide and the B-type peptide (hereinafter referred to as 'A-type phage' and 'B-type phage' for convenience), which were selected through the procedure of Example 2, were grown, followed by immobilization of human engineeriogenin. Bound to a 96-hole plate, the relative amount of bound phage was confirmed as follows.

100ng of human engiogenin was immobilized in a 96-hole ELISA container, phosphate buffer containing 1.5% albumin and 0.25% lysozyme was added and left at 37 ° C. for 4 hours, and then 10 ⁹ phages were added. The combined phages with engiogenin at 37 ° C. for 2 hours, and the non-stick phages were removed by washing 5 times with phosphate buffer containing 0.1% Tween 20.

Subsequently, both antibodies (Pharmacia Biotech, USA) against M13 phage diluted to 1 / 1,000 were treated at 37 ° C. for 1 hour, washed three times with phosphate buffer containing 0.1% of Tween 20, and then 1 / 1,500 The antibodies to both antibodies bound to the peroxidase diluted with were treated at 37 ° C. for 1 hour. After washing three times with phosphate buffer containing 0.1% Tween 20, it was treated with o-phenyldiaminedihydrochloride (OPD), an aqueous substrate of peroxidase. After a certain period of time, the reaction was stopped with 3N hydrochloric acid, and the absorbance was measured at a wavelength of 490 nm to determine the amount of phages bound to angiogenin.

Figure 2 shows that type A and B phage bind well to the plate on which the geogenin is fixed, but hardly adhere to the plate (control) to which the geogenin is not attached. In addition, it can be seen that the phage library itself used for the search is hardly bound with the engiogenin. Therefore, it was confirmed that the binding between the A-type and B-type phage and the engiogenin occurred through the peptide expressing phage.

Figure 3 shows the relative amounts of phage A and B phages treated with a reducing agent (1 mM DTT), protease (Factor Xa) or RNase (50 μM) at room temperature for 30 minutes, followed by the above-mentioned method. One result. At this time, the inhibition rate was expressed as a percentage of the control group not treated with the reducing agent, protease or RNase. As shown in Figure 3, it was found that the treatment with the reducing agent inhibited the binding to engiogenin by about 90%, and by the protease, about 70%.

These results indicate that, as shown in FIG. 1, the specific peptide expressed by each phage constituting the phage library is restricted by disulfide bonds and is linked to the pIII protein through the recognition amino acid sequence of a specific protease (Factor Xa). Therefore, it has been suggested that the binding between the A-type and B-type phage and the enzygenin occurs through the peptide expressed by the phage, and this indicates that the limited structure by disulfide bonds is important. In addition, RNase, a protein similar to engiogenin, does not affect the binding between type A and type B phages and engiogenin even in the presence of 50 μM, indicating that the binding of engiogenin to type A and B phages is specific to Could know.

4 shows that the binding of A and B phages to angiogenin is concentration-dependently inhibited by actin, an angiogenin receptor. In addition, FIG. 5 shows that the binding of A and B phages to engiogenin is concentration-dependently inhibited by the synthesized ANI-E peptide. The ANI-E peptide was derived from the amino acid sequence of the A-type peptide and was synthesized in a restricted form by disulfide bonds (see Example 4 below).

From the above results, the A-type and B-type phages are specifically bound to angiogenin, and the binding is made by a specific peptide expressed by the phage, and at this time, a limited structure by disulfide bonds is important, and It was found that the binding was inhibited by actin, an engineering enzyme.

Example 4

[Synthesis and Purification of ANI-E and ANI-V Peptides]

ANI-E peptides derived from type A peptides (NH ₂ -Ala-Gln-Leu-Ala-Gly-Glu-Cys-Arg-Glu-Asn-Val-Cys-Met-Gly-Ile-Glu-Gly-Arg- COOH) and an ANI-V peptide (NH ₂ -Ala-Gln-Leu-Ala-Gly-Glu-Cys-Arg-Val-Asn-), a mutation of ANI-E in which glutamic acid between two cysteines is substituted with valine Val-Cys-Met-Gly-Ile-Glu-Gly-Arg-COOH) was chemically synthesized using a peptide synthesizer (Peptido Genic Research & Co., USA) in a form restricted by disulfide bonds.

The synthesized peptide was injected into a C ₁₈ reverse phase column (Bio-Synthesis, Inc., USA) for high performance liquid chromatography, and solvent A (distilled water / 0.05% trifluoroacetic acid) and solvent B (acetonitrile / 0.1% trifluoroacetic acid). Purification was carried out using acetonitrile gradient of 10% to 70%. Purified ANI-E and ANI-V peptides were dissolved in distilled water at a concentration of 5 mg / ml, dispensed in appropriate amounts and stored at -70 ° C.

Example 5

[Analysis of binding inhibition between angiogenin and its receptor actin by ANI-E peptide]

In order to determine whether the ANI-E peptide synthesized and purified in Example 4 inhibits the binding between human angiogenin and its receptor actin, the following experiment was carried out. That is, 10ng of human engiogenin was fixed in a 96-hole ELISA container, and a phosphate buffer solution containing 1% albumin and 1% gelatin was added thereto and left at 37 ° C. for 4 hours. Subsequently, actin (1.6nM) labeled with biotin was bound to the imogenogenin immobilized in the absence or presence of peptide for 1 hour at 37 ° C. Unbound actin 0.1% of Tween 20. The washing three times with the phosphate buffer solution, and 58,000cpm labeled with I ¹²⁵ in order to determine the amount of an actin-binding and NGO Janine streptavidin (20μCi / μg, Amersham, USA), unbound streptavidin was washed three times with phosphate buffer containing 0.1% Tween 20, and then bound streptavidin was liberated with 0.1 N sodium hydroxide. The amount was measured with a sweetness meter.

As a result, the ANI-E peptide inhibited the binding between human angiogenin and actin in a concentration-dependent manner and inhibited 50% at 1 μM concentration. When the ANI-E peptide was treated with a reducing agent (1 mM DTT), the binding ability of the peptide was decreased. Significantly decreased, it was also found that the binding inhibition of the ANI-V peptide, which is a mutant form of the ANI-E peptide, was almost insignificant (see FIG. 6).

Therefore, ANI-E peptides derived from type A phage inhibit the binding between angiogenin and actin, where glutamic acid between the restricted structure and cysteine by disulfide bond plays an important role in the activity of ANI-E peptide. It was.

Example 6

[Ane-E Peptide Inhibition of Renal Angiogenesis Induced by Engiogenin]

Experiments using an egg chorioallantoic membrane (CAM) examined whether the ANI-E peptide inhibited renal angiogenesis induced by human engiogenin. In other words, put the fertilized egg (Pulmuone Co., Ltd.) into a 37 ° C incubator with 90% humidity, incubate for 3 days, remove 2 ml of albumin, and remove eggshells of 2cm × 2cm after 4 days. After incubation, the culture was continued. 10 ng of human engineeriogenin and peptide or actin are mixed, 3 μl is dipped into 1/4 piece of Thermanox disc (Nunc, USA) and dried, followed by incubation for 2 days on CAM of 9-day embryos, followed by instilled sample Whether or not there were blood vessels to be determined was determined by two people irrespective of each other under a dissecting microscope. The experiment was conducted with 10-20 eggs per each sample and repeated 4 times.

As described above, the results of quantitative analysis of the effects of ANI-E peptide on renal angiogenesis induced by angiogenin are shown in Table 3 below.

As shown in Table 3 above, in the presence of 10 ng of engiogenin, renal angiogenesis was induced in 45.7% of eggs, but in the absence of engiogenin, renal angiogenesis was induced in only 17.7% of eggs. It is well induced renal angiogenesis. In addition, angiogenin-induced renal angiogenesis was observed to be concentration-dependently inhibited by the ANI-E peptide, which was inhibited by 50% when the peptide was present 28-fold more than angiogenin. All of the renal angiogenesis was inhibited. However, the ANI-E peptide itself did not affect the angiogenesis produced during egg embryonic development regardless of concentration. Actin, an acceptor of angiogenin, inhibited neovascularization induced by angiogenin by 60% when present at 20-fold. On the other hand, the mutated ANI-V peptide hardly inhibited renal angiogenesis induced by engiogenin even in the presence of 285-fold excess. This result is in good agreement with the result that the ANI-V peptide did not inhibit the binding of angiogenin to its receptor actin (see Example 5).

Therefore, the ANI-E peptide selectively inhibits renal angiogenesis induced by angiogenin, and according to the results of Examples 3 to 6, the mechanism of inhibiting angiogenesis of the ANI-E peptide was It was predicted to inhibit binding between actin, a receptor.

Example 7

[Ane-E Peptide Inhibition of Renal Angiogenesis Induced by Cancer Cells Secreting Engiogenin]

Experiments with egg CAM confirmed that the ANI-E peptide inhibited renal angiogenesis induced by cancer cells secreting human engiogenin. In the same manner as in Example 6, the fertilized eggs were incubated at 37 ° C. to remove albumin and to form windows. Separately, 10 ⁵ human prostate cancer cells (PC 3, human prostate adenocarcinoma (ATCC CRL-1435), 7.5 μg of Type I collagen (Rat tail, Beckton Dickinson, USA) and ANI-E peptide were mixed in a volume of 5 μl, 1 Collagen sponges were prepared at room temperature by dropping onto / 4 pieces of Thermanox discs. This collagen sponge was placed on the egg CAM of 10 days and incubated at 37 ° C. for 3 days, and then blood vessels induced by the inoculated cancer cells were confirmed in the same manner as in Example 6. All experiments were conducted simultaneously using 12 eggs for each sample.

As a result, in the presence of prostate cancer cells, renal angiogenesis was induced from the surrounding blood vessels toward the collagen sponge, but this phenomenon was not observed in the collagen sponge without prostate cancer cells. In addition, ANI-E peptide (258 pmol) inhibited renal angiogenesis induced by prostate cancer cells without affecting existing blood vessels. In contrast, the ANI-V peptide (262 pmol) did not inhibit renal angiogenesis induced by cancer cells, which is in good agreement with the results of Examples 5 and 6.

In order to quantitatively analyze the degree of inhibition of angiogenesis, the number of renal vessels induced by cancer cells is shown in FIG. 7 under the microscope. Prostate cancer cells without peptide were induced 42 angiogenesis (standard deviation = 7) towards collagen sponge (control), whereas ANI-E peptide (258 pmol) inhibited 64% of neovascularization induced by cancer cells. , Mutated ANI-V peptide (262 pmol) inhibited only about 10%.

Therefore, ANI-E peptide inhibits angiogenin secreted by prostate cancer cells, thereby inhibiting angiogenesis induced by cancer cells, but does not affect existing blood vessels, and thus can be useful as a cancer treatment agent without side effects. Could.

Example 8

[A direct effect assay of ANI-E peptide on the growth of human prostate cancer cells]

Whether the ANI-E peptide directly affects the growth of prostate cancer cells was examined as follows. PC 3 cells (7 × 10 ³ ) were placed in a culture vessel (96well, Nunc, USA) and cultured for one day, and medium containing various concentrations of ANI-E peptide (RPMI 1640 + 10% fetal calf serum) was added. . After incubation for 3 days, 20 μl tetrazolium dye (Cell Titer 96 Non-Radioactive Proliferation assay kit, Promega, USA) was added and incubated at 37 ° C. for 4 hours, and the formazan amount produced by living cells was 0.2 ml of lysis solution. (Promega, USA) was lysed, and the absorbance was measured at 570 nm to determine the number of living cells (see FIG. 8). At this time, the absorbance by the lysed formazan is proportional to the number of living cells.

As shown in Figure 8, the ANI-E peptide did not affect the growth of PC 3 cells even at a concentration of about 10 ^-4 M.

From Examples 7 and 8, it can be seen that the ANI-E peptide specifically inhibits angiogenesis induced by prostate cancer cells without directly affecting the growth of prostate cancer cells, which indicates that the ANI-E peptide prostate This means that angiogenin secreted by cancer cells inhibits binding to actin, which is a receptor thereof, thereby inhibiting renal angiogenesis induced by prostate cancer cells.

As described and demonstrated in detail above, the peptide of the present invention concentration-dependently inhibits the binding between human angiogenin and its receptor actin, and eventually does not affect the existing blood vessels in renal angiogenesis induced by human engiogenin. Inhibition may be effectively used as an anticancer agent or a therapeutic agent for diseases such as rheumatoid arthritis and diabetic blindness in which nephrogenic angiogenesis is essential.

Claims (4)

Two cysteine disulfide-linked peptides consisting of the following amino acid sequences that inhibit angiogenesis by inhibiting the binding of angiogenin to its receptor actin: NH ₂ -Ala-Gln-Leu-Ala-Gly-Glu-Cys-Arg-Glu-Asn-Val-Cys-Met-Gly-Ile-Glu-Gly-Arg-COOH The peptide according to claim 1, wherein the inhibition of binding between the enzyme and actin is by direct binding between the peptide and the enzyme. The peptide of claim 1, wherein the peptide inhibits renal angiogenesis induced by engiogenin without affecting existing blood vessels. A method of inhibiting renal angiogenesis comprising administering the peptide of claim 1 to a mammal, except for a person with abnormally sustained renal angiogenesis.

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